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EP0585168A2 - Zusammensetzung und Verfahren zur Knochenerzeugung - Google Patents

Zusammensetzung und Verfahren zur Knochenerzeugung Download PDF

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Publication number
EP0585168A2
EP0585168A2 EP93402073A EP93402073A EP0585168A2 EP 0585168 A2 EP0585168 A2 EP 0585168A2 EP 93402073 A EP93402073 A EP 93402073A EP 93402073 A EP93402073 A EP 93402073A EP 0585168 A2 EP0585168 A2 EP 0585168A2
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EP
European Patent Office
Prior art keywords
bone
tgf
composition
dbm
implants
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP93402073A
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English (en)
French (fr)
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EP0585168A3 (en
Inventor
A. Gregory Bruce
D. Michael Strong
Douglas J. Kibblewhite
Wayne R. Gombotz
Wayne F. Larrabee
Anthony F. Purchio
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Bristol Myers Squibb Co
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Bristol Myers Squibb Co
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Publication of EP0585168A2 publication Critical patent/EP0585168A2/de
Publication of EP0585168A3 publication Critical patent/EP0585168A3/en
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3608—Bone, e.g. demineralised bone matrix [DBM], bone powder
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1841—Transforming growth factor [TGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K6/00—Preparations for dentistry
    • A61K6/80—Preparations for artificial teeth, for filling teeth or for capping teeth
    • A61K6/884—Preparations for artificial teeth, for filling teeth or for capping teeth comprising natural or synthetic resins
    • A61K6/887—Compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K6/00—Preparations for dentistry
    • A61K6/80—Preparations for artificial teeth, for filling teeth or for capping teeth
    • A61K6/884—Preparations for artificial teeth, for filling teeth or for capping teeth comprising natural or synthetic resins
    • A61K6/891—Compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
    • A61L27/3645—Connective tissue
    • A61L27/365—Bones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • A61L2300/414—Growth factors

Definitions

  • the present invention is directed to a composition and a method of generating useful, non-resorptive bone formation.
  • the present invention can be utilized for the treatment of fractures, skeletal defects and maxillofacial reconstructions.
  • Bone has a remarkable regenerative capacity; however, adverse conditions may affect the bony repair process promoting fibrogenesis at the expense of bone union.
  • maxillofacial and orthopedic surgeons employ osteoinductive agents such as grafts and implants to restore continuity and contour defects which arise from trauma, infection and neoplasm.
  • autologous cancellous bone is the choice graft material employed for both craniomandibulofacial and long bone defects.
  • Osteoinduction may be enhanced by combining autogenetic or allogenic bone with osteogenic progenitor cells from bone marrow (Lindholm T.S. et al. (1982) Clin. Orthop. 171 :251; Simmons, D.J. et al. (1973) Clin.
  • DBM demineralized bone matrix
  • Non-osseous tissues are also capable of responding to these osteoinductive growth factors including connective tissue such as muscle and muscle fascia (Urist M.R. et al. (1969) Clin. Orth. 64 :194), thymus (Friedenstein A.J. (1973) Determined and Inducible Osteogenic Precursor Cells In Hard Tissue Growth, Repair and Remineralization. Sognas R. and Vaughan J. (eds). Ciba Foundation Symposium II. Associated Scientific Publishers, Amsterdam, p. 169) and the connective tissue sheaths of the visceral organs (Chalmers J. et al. (1975) J. Bone Joint Surg. 57B :36).
  • connective tissue such as muscle and muscle fascia (Urist M.R. et al. (1969) Clin. Orth. 64 :194), thymus (Friedenstein A.J. (1973) Determined and Inducible Osteogenic Precursor Cells In Hard Tissue Growth, Repair and
  • Cytodifferentiation of uncommitted mesenchymal cells involves a sequential series of events: mesenchymal cell proliferation and activation, cartilage formation, woven bone formation and finally, generation of lamellar bone.
  • mesenchymal cell proliferation and activation mesenchymal cell proliferation and activation
  • cartilage formation a sequential series of events
  • woven bone formation a sequential series of events
  • lamellar bone a progressive osteogenic protein
  • transforming growth factor-beta transforming growth factor-beta
  • Demineralized bone has been used in a variety of craniomandibulofacial applications, however, the amount of actual new bone growth has varied considerably (Mulliken J.B. (1985) Induced Osteogenesis and Craniofacial Surgery In Caronnie E. (ed.): Craniofacial Surgery. Little Brown, Boston; Toriumi D.M. et al. (1990) Arch. Otolaryngol. Head Neck Surg. 116 :676). High resorption rates occur when demineralized bone is used in augmentation procedures over the nasal dorsal, malar eminence, supraorbital ridge, nasal tip and along the mandible.
  • the present invention has placed emphasis on the isolation, characterization and purification of matrix-bound protein growth factors responsible for osteoinduction.
  • Several structurally related bone growth factors have now been isolated and sequenced (Wang E.A. et al. (1988) PNAS USA 85 :9484; Wang E.A. et al. (1990) PNAS USA 85 :2220; Ozkaynak E. et al. (1990) EMBO 9 :2085), combined with biodegradable carriers for delivery and tested in experimental animal models.
  • biodegradable carriers for delivery and tested in experimental animal models.
  • the bone repair rate among experimental animals varies inversely with order along phylogenetic scale (Prolo, D.J. et al. (1982) Clin. Orthop. 108 :230) and with the maturity (age) of the animal (Prolo, D.J. et al. (1982) Clin. Orthop. 108 :230; Robinson R.A. (1971) J. Bone Joint Surg. 53A :1017).
  • the majority of available osteoinductive proteins have been shown to have excellent bone forming capabilities when used in low order, immature species, having unlimited capacity for spontaneous healing.
  • the quality of bone repair also depends on the anatomic location (Harris W.H. et al. (1968) J. Bone Joint Surg.
  • a critical size defect heals by fibrous obliteration (Frame J.W. (1980) J. Oral Surg. 38 :176) and not bone formation and is currently the best prototype available to study the process of maxillofacial and mandibular nonunion (Schmitz J.P. et al. (1986) Clin. Orthop. Rel. Res. 205 :299).
  • the calvarial critical size defect is considered the most severe test of osteoinductive capacity because the cranium is biologically relatively inert; corresponding to a deficiency in marrow and an absent nutrient blood supply (Prolo D.J. et al.
  • TGF- ⁇ is a 24,000 Dalton polypeptide which modulates the growth and differentiation of many cell types.
  • the latent TGF ⁇ in bone comprises the largest pool in the body by a factor of ten. It is a highly conserved protein, which suggests an essential role in cell physiology.
  • TGF- ⁇ The human and monkey mature forms of TGF- ⁇ are identical. Most human cell types studied express both TGF- ⁇ and receptors for it (Bonewald L.F. et al. (1990) Clin. Orthop. 250: 261-76). It is secreted in a latent form, bound to a large carrier protein. Its activation is the subject of intense investigation. Extremes in pH, and enzymes such as cathepsin D and plasmin have been identified as activators (Pfeilschifter J. et al. (J. Bone Mineral Res. 5(1) :49-58).
  • TGF- ⁇ stimulates osteoblast-like cells to markedly increase the production of type I collagen, osteonectin, osteopontin, and fibronectin
  • TGF- ⁇ inhibits osteoclasts, and osteoclasts activate bone-latent TGF- ⁇ .
  • the low pH and high enzyme activity found under the ruffled border of osteoclasts forms the ideal microenvironment for activation. Since osteoclasts activity is the primary event in bone remodeling, this could provide the trigger for activation of bone-latent TGF- ⁇ with subsequent osteoclast stimulation and new bone matrix production (Bonewald L.F. et al. (1990) Clin. Orthop. 250 :261-76).
  • DBM and a TGF- ⁇ are combined to generate new bone which is capable of meeting the criteria of the CSD model and which overcomes the problems of bone resorption previously resultant from the use of DBM.
  • New, hard, non resorptive bone is produced in this invention.
  • the present invention is directed to compositions and methods for facilitating the growth and formation of hard, non-resorptive bone.
  • Compositions of the present invention containing demineralized bone matrix and TGF- ⁇ can be utilized to stimulate the development of bone in areas of the body where new bone formation is desired.
  • compositions and methods of this invention can be utilized for therapeutic, cosmetic, and surgical treatments ranging from osteoporosis to mandibular reconstruction to treatment of non-union bone fractures.
  • the present invention is directed to a method for inducing the production of hard, non-resorptive bone growth.
  • Bone growth is stimulated in this invention by a composition containing a demineralized bone matrix and a transforming growth factor beta.
  • BMP-2 bone morphogenic protein
  • DBM demineralized bone matrix
  • the DBM has low levels of growth factor activity.
  • the factors detected in DBM are listed in TABLE 1.
  • the DBM is operatively attached to a TGF- ⁇ .
  • DBM is combined with a sufficient amount of TGF- ⁇ to result in an essentially irreversible binding of TGF- ⁇ to the DBM.
  • the DBM of the present invention is a bone derivative prepared by demineralizing bone with HCl, followed by lyophilization.
  • the final powder is composed of particles ranging in size from 74 ⁇ m to 640 ⁇ m.
  • TGF- ⁇ is a 24 kD homodimeric protein that stimulates the migration, proliferation and matrix synthesis of mesenchymal cells. Bone is the largest reservoir of TGF- ⁇ in the body (S.M. Seyedin et al. (1986) J. Biol. Chem. 261 :5693-5695) , and its ability to promote bone formation suggest that it may have potential as a therapeutic agent in diseases of bone loss (Noda, M. and Camilliere, J. (1989) Endocrinology 124 :2991-2207). The in vivo application of TGF- ⁇ adjacent to periosteum has been shown to increase bone thickness and chondrogenesis (Joyce et al. (1990) J. Cell Biol.
  • TGF- ⁇ has also been shown to induce bone closure of large bony defects in the skull (Beck et al. (1991) J. Bone and Miner. Res. 6 :1257-1265).
  • the composition of TGF- ⁇ and DBM can be utilized for the treatment of fractures, in dental implants and for the treatment of structural defects, such as in maxillofacial reconstructions.
  • Particularly useful embodiments of the present invention comprise the application of a formed matrix containing DBM and TGF- ⁇ to a body area where bone growth of a specified shape or form is desired.
  • a composition of this invention can be shaped and fitted in a patient to produce bone growth of a mandibular region of the jaw in a patient needing reconstructive surgery as a result of head and neck trauma or disease.
  • the present invention can also encompass compositions and implants for the treatment of dental disorders, such as tooth loss and bone defects, (referred to herein as "dental implants").
  • a particularly useful composition of the present invention is present in a non-fluid, plastic malleable matrix which can be form-fitted to a site where bone growth is desired. New bone formation can then occur according to the shape and form of the matrix.
  • This method can produce new bone which would resemble and replace or repair bony structures for reconstructive surgery, such as jaws, skull plates, cheek bones, collar bones, non-union fractures, fingers, toes, etc.
  • TGF- ⁇ refers to any member of the family of transforming growth factor beta which include TGF- ⁇ 1, TGF- ⁇ 2, TGF- ⁇ 3, TGF- ⁇ 4, TGF- ⁇ 5 as well as the TGF- ⁇ 1/ ⁇ 2 hybrid molecules, designated TGF-5 ⁇ .
  • TGF-5 ⁇ A novel form of TGF- ⁇ , designated TGF-5 ⁇ , comprises a hybrid amino acid residue sequence of TGF- ⁇ 1 and TGF- ⁇ 2. TGF-5 ⁇ has more effective recombinant processing and expression than has been seen for either TGF- ⁇ 1 or TGF- ⁇ 2. Specific description of TGF-5 ⁇ is disclosed in Madisen et al (1990) in Transforming Growth Factor ⁇ 's. Chemistry. Biology and Therapeutics , Anal. N.Y. Acad. Sci. 593 :7-25 and in U.S. patent application serial numbers 669,171, filed March 14, 1991, and 667,246, filed March 8, 1992, the disclosures of which are incorporated herein by reference.
  • unit dose and "sufficient amount” and grammatical variations thereof, are used interchangeably and refer to physically discrete amounts of material, such as TGF- ⁇ and DBM, suitable as unitary dosages for patients, each unit contains a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; that is, a carrier or vehicle.
  • the specifications for the novel unit dose of this invention are dictated by, and are directly dependent upon, (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such active material for therapeutic use.
  • the term "effective amount” means an amount sufficient to stimulate bone production and formation at the desired site in a patient.
  • the effective amount for a particular patient may vary depending on such factors as the extent of the fracture, injury or defect, the overall health of the patient, the method of administration, the severity of any side effects, and the like.
  • conservative substitution denotes that one amino acid residue has been replaced by another, biologically similar residue.
  • conservative substitutions include the substitutions of one hydrophobic residue such as Ile, Val, Leu, or Met for another, or the substitution of one polar residue for another such as between Arg and Lys, between Glu and Asp or between Gln and Asn, and the like.
  • an ionic residue by an oppositely charged ionic residue such as Asp by Lys has been determined conservative in the art in that those ionic groups are thought to merely provide solubility assistance.
  • replacements discussed herein are on a relatively short synthetic peptide region, as compared to a whole protein, replacement of an ionic residue by another ionic residue of opposite charge is considered herein to be a "radical replacement" as are replacements by nonionic and ionic residues, and bulky residues such as Phe, Tyr or Trp and less bulky residues such as Gly, Ile and Val.
  • nonionic and ionic residues are used herein in their usual sense to designate those amino acid residues that either bear no charge or normally bear a charge, respectively, at physiological pH value.
  • exemplary nonionic residues include Thr and Gln, while exemplary ionic residues include Arg and Asp.
  • Ferric salts of the present invention encompass any physiologically tolerable organic salts and particularly includes ferric citrate.
  • phrases "pharmaceutically acceptable salts” and “physiologically tolerable salts”, as used interchangeably herein, refer to non-toxic alkali metal, alkaline earth metal and ammonium salts used in the pharmaceutical industry, including the sodium, potassium, lithium, calcium, magnesium and ammonium salts and the like that are prepared by methods well-known in the art.
  • the phrase also includes non-toxic acid addition salts that are generally prepared by reacting the compounds in this invention with a suitable organic or inorganic acid.
  • Representative salts include the hydrochloride, hyrdrobromide, sulfate, bisulfate, acetate, oxalate, valerate, oleate, laureate, vorate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate and the like.
  • the term "operatively attached” refers to the linkage of groups in a manner such that the desired biological activity of the group is not inhibited by the attachment.
  • operatively linked refers to a linkage that does not interfere with the ability of either of the linked groups to function as described.
  • DBM is operatively linked to a biologically active TGF- ⁇ compound in a manner that permits the TGF- ⁇ to interact with osteoblasts to stimulate bone formation in association with DBM and which does not interfere with the biological activity of the TGF- ⁇ . This allows TGF- ⁇ to be released when the DBM is absorbed by osteoclasts.
  • the TGF- ⁇ and DBM of the present invention are preferably present in a pharmaceutical composition together with one or more pharmaceutically acceptable carriers.
  • physiologically tolerable and “pharmaceutically acceptable” are used interchangeably and refer to molecular entities and compositions that do not produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a patient.
  • a carrier is a material useful for administering the active compound and must be "acceptable” in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof.
  • pharmaceutically acceptable carrier refers to a compound which is compatible with administration to a patient and does not produce toxic or untoward effects upon such administration.
  • pharmaceutically acceptable carriers are phosphate buffered saline, Ringer's solution, oils, gels, biodegradable matrices, including natural or synthetic polymers, and microspheres, as well as liposomes.
  • Other pharmaceutically acceptable carriers are well known in the field of pharmacy and are contemplated by the present invention.
  • labeling agent refers to a single atom or molecule that when operatively linked to a product of the present invention enables detection of its presence in an assay method.
  • Illustrative labeling agents are fluorescent dyes, radioisotopes, enzymes and antibodies that can be either independently detected or detected in conjunction with the addition of a substrate or other molecule that reacts therewith.
  • substantially refers to a high level of similarity.
  • a substantially purified peptide of the present invention refers to a preparation having less than about ten percent extraneous peptides present.
  • a substantially similar sequence in the present invention has less than about ten percent variation with the reference sequence.
  • compositions are prepared by any of the methods well known in the art of pharmacy all of which involve bringing into association the active compound and the carrier therefor.
  • the agents utilized in the present invention can be administered in the form of conventional pharmaceutical compositions.
  • Such compositions can be formulated so as to be suitable for parenteral administration, or as implants.
  • the agents are typically dissolved or dispersed in a physiologically tolerable carrier.
  • the compounds of the present invention can be utilized in non-fluid malleable, plastic compositions such as sterile suspensions or putty-like matrices capable of being formed to fit a fracture or defect, or as isotonic preparations containing suitable preservatives.
  • plastic compositions such as sterile suspensions or putty-like matrices capable of being formed to fit a fracture or defect, or as isotonic preparations containing suitable preservatives.
  • biodegradable malleable matrices constituted by aqueous isotonic and sterile semi-rigid materials which enable osteoblast migration into and bone formation in the shaped matrix
  • the pharmaceutical formulation described herein can include, as appropriate, one or more additional carrier ingredients such as diluents, buffers, binders, surface active agents, thickeners, lubricants, preservatives (including antioxidants) and the like, and substances included for the purpose of rendering the formulation isotonic with the blood of the intended recipient.
  • additional carrier ingredients such as diluents, buffers, binders, surface active agents, thickeners, lubricants, preservatives (including antioxidants) and the like, and substances included for the purpose of rendering the formulation isotonic with the blood of the intended recipient.
  • a temperature of about 20°C will include temperature values of from 18°C to 22°C.
  • plastic and “malleable” are used interchangeably and refer to material which can be formed or molded into specified shapes and which can be made to maintain the formed shape.
  • a particularly preferred support material of this invention comprises a biodegradable matrix containing DBM and TGF- ⁇ together with a degradable homopolymer or copolymer.
  • Illustrative homopolymers or copolymers include poly(lactic acid), poly(lactic-coglycolic acid), poly(glycolic acid), poly(caprolactone), poly(hydroxybutyric acid), poly(anhydrides), poly(orthoesters), poly(carbonates) poly(amides), poly(acetals), poly(amino acids) and poly(cyanoacrylates).
  • TGF- ⁇ 1 Transforming growth factor- ⁇ 1 (TGF- ⁇ 1) is a multifunctional regulator of cell growth and differentiation. It is capable of causing diverse effects such as inhibition of the growth of monkey kidney cells, (Tucker, R.F., G.D. Shipley, H.L. Moses & R.W. Holley (1984) Science 226 :705-707) inhibition of growth of several human cancer cell lines, (Roberts, A.B., M.A. Anzano, L.M. Wakefield, N.S. Roches, D.F. Stern & M.B. Sporn (1985) Proc. Natl. Acad. Sci. USA 82 :119-123; Collinsalis, J.E., L.E. Gentry, Y.
  • TGF- ⁇ 1 is a member of a family of closely related growth-modulating proteins including TGF- ⁇ 2, (Seyedin, S.M., P.R. Segarini, D.M. Rosen, A.Y. Thompson, H. Bentz & J. Graycar (1987) J. Biol. Chem. 262 :1946-1949; Cheifetz, S., J.A. Weatherbee, M.L.-S. Tsang, J.K. Anderson, J.E. Mole, R. Lucas & J. Massague (1987) Cell 48 :409-415; Ikeda, T., M.M. Lioubin & H.
  • TGF- ⁇ 1 is a 24-kDa protein consisting of two identical disulfide-bonded 12 kD subunits.
  • TGF- ⁇ 1 indicates that this protein is synthesized as a larger 390 amino acid pre-pro-TGF- ⁇ 1 precursor; the carboxyl terminal 112 amino acid portion is then proteolytically cleaved to yield the TGF- ⁇ 1 monomer.
  • the simian TGF- ⁇ 1 cDNA clone has been expressed to high levels in Chinese hamster ovary (CHO) cells. Analysis of the proteins secreted by these cells using site-specific antipeptide antibodies, peptide mapping, and protein sequencing revealed that both mature and precursor forms of TGF- ⁇ were produced and were held together, in part, by a complex array of disulfide bonds.
  • the pro-region of the TGF- ⁇ 1 precursor was found to be glycosylated at three sites (Asn 82, Asn 136, and Asn 176) and the first two of these (Asn 82 and Asn 136) contain mannose-6-phosphate residues.
  • the rTGF- ⁇ 1 precursor is capable of binding to the mannose-6-phosphate receptor and may imply a mechanism for delivery to lysosomes where proteolytic processing can occur.
  • TGF- ⁇ 2 is also a 24-kD homodimer of identical disulfide-bonded 112 amino acid subunits (Marquardt, H., M.N. Lioubin & T. Ikeda (1987) J. Biol. Chem. 262 :12127-12131).
  • Analysis of cDNA clones coding for human (Madisen, L., N.R. Webb, T.M. Rose, H. Marquardt, T. Ikeda, D. Twardzik, S. Seyedin & A.F. Purchio (1988) DNA 7 :1-8; DeMartin, R., B. Plaendler, R. Hoefer-Warbinek, H. Gaugitsch, M. Wrann, H.
  • TGF- ⁇ 2 showed that it, too, is synthesized as a larger precursor protein.
  • the mature regions of TGF- ⁇ 1 and TGF- ⁇ 2 show 70% homology, whereas 30% homology occurs in the pro-region of the precursor.
  • TGF- ⁇ TGF- ⁇ mediated by the binding to specific receptors present on the surface of most cells (Massague, J. et al. (1985) J. Biol. Chem. 260 :2636-2645; Segarini, P.R. et al. (1989) Mol. Endocrino. 3 :261-272; Tucker, R.F., et al. (1984) Proc. Natl. Acad. Sci. USA 81 :6757-6761; Wakefield, L.M., et al. (1987) J. Cell Biol. 105 :965-975).
  • TGF- ⁇ has been shown to induce fos , myc and sis in AKR-2B cells (Leof, E.B., et al. (1986) Proc. Natl. Acad. Sci. USA 83 :1453-1458):1453-1458) enhance expression of c- jun B in A549 cells (Pertovaara, L., et al. (1989) Molecular and Cellular Biology 9 :1255-1264), increase the mRNA for matrix proteins (Penttinen, R.P., et al. (1988) Proc. Natl. Acad. Sci.
  • TGF- ⁇ induces expression of IL-1 ⁇ , TNF- ⁇ , PDGF and bFGF in human peripheral blood monocytes (McCartney-Francis, N., et al. (1991) DNA and Cell Biology 10 :293-300).
  • compositions and methods of the present invention can be utilized in therapeutic and reconstructive processes of bone formation such as regeneration and supplementation of osteoporotic bone, facial skeletal augmentation, reconstruction of mandibular and craniofacial defects, and periodontal applications.
  • the present invention contemplates a composition and method for the stimulation of growth of new bone and the regeneration of bone in fractures, repair of acetabular defects, osteoporosis, fillings for benign cystic lesions or tumor lesions of bone, and skeletal defects.
  • Implants containing compositions of DBM and TGF- ⁇ , prepared under sterile conditions, can be implanted at bony sites where bone formation is desired. Sites at which such bone formation is contemplated include bone fracture, mandibular defects, cranial holes produced during surgery, tooth fillings and periodontal defects.
  • the bone formed in the present inventions is vascularized and ossified, and is indistinguishable from naturally produced bone in structure and function.
  • the bone formed in the present invention is not resorbed in contrast to prior bone formation stimulated by bone powder.
  • the combination of DBM with TGF- ⁇ was found by the present inventors to produce hard, functional non-resorptive bone which is useful in the treatment of fractures, periodontal disorders and skeletal defects requiring the formation or regeneration of bone.
  • the methods and compositions of this invention are contemplated to be useful in plastic surgery, maxillofacial reconstruction, treatment of fractures, bone loss and skeletal defects and deformities.
  • the osteogenic compositions containing DBM operatively attached to TGF- ⁇ is preferred to be in an embodiment which is plastic and malleable and capable of being formed into shapes and designs conducive to the above applications and uses.
  • One contemplated use of these compositions is for the restructuring and reshaping of mandibular bone following maxillofacial reconstruction .
  • Another contemplated use is for the treatment of non-union fractures.
  • a third contemplated use is in dental implants for the production or regeneration of new bone and for the attachment of dental fixtures to the jaw. All uses consistent with the spirit and scope of the present invention are contemplated herein.
  • TGF- ⁇ is operatively linked to a carrier or matrix component, such as hydroxyapatite, in the composition with DBM.
  • a carrier or matrix component such as hydroxyapatite
  • the TGF- ⁇ remains localized in the support matrix and bone formation is stimulated as a result of osteoblast interaction with DBM and the immobilized TGF- ⁇ .
  • Demineralized bone matrix was prepared under sterile conditions from the harvested long bones of exsanguinated mature New Zealand White (NZW) rabbits. The bones were crushed, and cleaned by gentle agitation in warm sterile water and dried. The cleaned bones were then ground in a Tekmar bone mill to a particle size of 74 to 565 ⁇ m, assured by sieving. The ground bone was demineralized by repeated mixing with 0.6 M HCl, until no pH change occurred upon further washing with 0.6 M HCl. The pH was then adjusted to pH 7.27 with phosphate buffered saline, and the ground bone was lyophilized.
  • the processed DBM remained sterile. Repeated cultures of the DBM during processing were negative for bacteria and limulus endotoxin testing of the final product was negative.
  • Implants were prepared by reconstituting 300 mg DMB with 0.7 ml of 1 % rabbit serum albumin (Sigma Chemical Co., St. Louis, MO) in PBS to provide a thick paste. Either recombinant TGF- ⁇ 1 or carrier solution was then added to the paste. Implants were prepared containing 0, 1, 10, 100 and 250 ⁇ g, respectively, of TGF- ⁇ 1 per gram dry weight of DBM. The implants were shaped into cylinders approximately 7 mm in diameter and 25 mm long.
  • rabbit serum albumin Sigma Chemical Co., St. Louis, MO
  • the rabbits were divided into four groups which received implants containing 1, 10, 100 and 250 ⁇ g/gram dry weight of DBM, respectively. Under sterile operating room conditions, anesthesia was administered to the rabbits by intramuscular injection of ketamine and xylazine. A 1 cm incision was made in the supraorbital area, and a subcutaneous pocket corresponding to the dimensions of the implant was dissected immediately above the periosteum, which was lightly abraded with a rasp. The implant was placed on the periosteum and the incision was closed. The 1 ⁇ g group received a second implant without TGF- ⁇ 1 as a control.
  • the animals were fed a normal diet and housed in wire cages. Triple tetracycline labeling of bone formation was performed by administering intramuscular injections of tetracycline hydrochloride (20 mg/kg, Lederle Labs, Pearl River, NJ), demeclocycline (15 mg/kg, Lederle labs, Pearl River, NJ) and tetracycline hydrochloride (20 mg/kg) on days 16, 27 and 38, respectively.
  • the animals were sacrificed at day 42 by intracardiac pentobarbital overdose.
  • the implants were harvested intact in continuity with the underlying cranium.
  • the isolated samples were tagged and placed in iced 10% buffered formaldehyde solution.
  • TGF- ⁇ 1/DBM implants containing 250 ⁇ g TGF- ⁇ /g DBM were incubated in PBS at 37° C while shaking. At specified times, the buffer was removed and replaced with a new buffer. The amount of TGF- ⁇ 1 released from the implants into the buffer was determined by ELISA.
  • Figure 4 shows the amount of TGF- ⁇ 1 released over time from two different implants.
  • One implant was prepared by mixing the DBM with TGF- ⁇ 1 in a citrate buffer (pH 2.5) while other was made with a PBS buffer (pH 7.4). In both samples the most significant release of TGF- ⁇ 1 occurred over the first 120 hours.
  • the sample prepared with citrate buffer released approximately 10% of the TGF- ⁇ while the sample prepared in PBS released about 7%.
  • FIGURES 6A and 6B A second in vitro release study was done with DBM implants containing different concentrations of TGF- ⁇ 1 and TGF-5 ⁇ . The release kinetics are shown in FIGURES 6A and 6B. Again, only a small percentage of the TGF- ⁇ that is bound to the DBM is released. A greater percentage of the TGF- ⁇ 1 is released from the 1250 ⁇ g TGF- ⁇ 1/g DBM implants than from the samples containing lower loadings of TGF- ⁇ 1. The implants containing TGF-5 ⁇ exhibit a similar trend although both the 1250 and 250 ⁇ g TGF-5 ⁇ g DBM samples release a greater percent of TGF-5 ⁇ than the 100 ⁇ g TGF-5 ⁇ /g DBM sample. These samples were also extracted in 4 M guanidine hydrochloride. FIGURE 7 shows that more TGF-5 ⁇ was extracted from the DBM than TGF- ⁇ 1. These results may indicated differences in DBM binding affinities between TGF- ⁇ 1 and TGF-5 ⁇ .
  • TGF- ⁇ 1 or TGF-5 ⁇ is released from DBM implants under physiological conditions, while more TGF- ⁇ is released from implants prepared with a citrate buffer (pH 2.5) than from implants prepared with PBS (pH 7.4).
  • a greater percentage of TGF-5 ⁇ can be released from or extracted from DBM than TGF- ⁇ 1, suggesting that TGF- ⁇ 1 is more tightly bound to DBM.
  • the TGF- ⁇ 1 remains stable in the DBM when stored at 4° C for up to one month.

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WO1998014222A1 (en) * 1996-09-30 1998-04-09 Children's Medical Center Corporation Methods and compositions for programming an organic matrix for remodeling into a target tissue
US6165487A (en) * 1996-09-30 2000-12-26 Children's Medical Center Corporation Methods and compositions for programming an organic matrix for remodeling into a target tissue
EP1113072A1 (de) * 1999-12-28 2001-07-04 Isotis B.V. Gewebetechnologie mit Zellen aus dem Kiefer
WO2002032474A1 (en) * 2000-10-13 2002-04-25 Osteotech, Inc. Method for inducing new bone growth in porous bone sites
WO2002069988A2 (en) * 2001-03-05 2002-09-12 Hadasit Med Res Service Compositions comprising bone marrow cells together with demineralized and/or mineralized bone matrix and their use in bone marrow transplantation
US6649168B2 (en) 1999-03-17 2003-11-18 Novartis Ag Pharmaceutical compositions comprising TGF-beta
WO2008022182A1 (en) * 2006-08-16 2008-02-21 The Uab Research Foundation Methods for promoting coupling between bone formation and resorption
US7910554B2 (en) 2001-04-12 2011-03-22 Bioaxone Therapeutique Inc. Treatment of macular degeneration with ADP-ribosyl transferase fusion protein therapeutic compositions
US7981156B2 (en) * 1995-10-16 2011-07-19 Warsaw Orthopedic, Inc. Bone grafts
USD849946S1 (en) 2015-12-30 2019-05-28 Nuvasive, Inc. Interspinous process spacer

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WO1989004646A1 (en) * 1987-11-13 1989-06-01 Jefferies Steven R Bone repair material and delayed drug delivery
EP0419275A1 (de) * 1989-09-21 1991-03-27 Osteotech, Inc., Zusammensetzung, die ein fliessfähiges entmineralisiertes Knochenpulver enthält und ihre Verwendung bei der Knochenheilung
WO1992013565A1 (en) * 1991-01-31 1992-08-20 Shaw, Robert, Francis Growth factor containing matrix for the treatment of cartilage lesions
EP0567391A1 (de) * 1992-04-24 1993-10-27 Bristol-Myers Squibb Company Biologisch abbaubarres TGF-Beta-Abgabesystem für Knochenregenerierung

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EP0169016A2 (de) * 1984-07-16 1986-01-22 Celtrix Pharmaceuticals, Inc. Polypeptide induzierende Faktoren in Knochen und Knorpel
WO1989004646A1 (en) * 1987-11-13 1989-06-01 Jefferies Steven R Bone repair material and delayed drug delivery
EP0419275A1 (de) * 1989-09-21 1991-03-27 Osteotech, Inc., Zusammensetzung, die ein fliessfähiges entmineralisiertes Knochenpulver enthält und ihre Verwendung bei der Knochenheilung
WO1992013565A1 (en) * 1991-01-31 1992-08-20 Shaw, Robert, Francis Growth factor containing matrix for the treatment of cartilage lesions
EP0567391A1 (de) * 1992-04-24 1993-10-27 Bristol-Myers Squibb Company Biologisch abbaubarres TGF-Beta-Abgabesystem für Knochenregenerierung

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7981156B2 (en) * 1995-10-16 2011-07-19 Warsaw Orthopedic, Inc. Bone grafts
US6165487A (en) * 1996-09-30 2000-12-26 Children's Medical Center Corporation Methods and compositions for programming an organic matrix for remodeling into a target tissue
WO1998014222A1 (en) * 1996-09-30 1998-04-09 Children's Medical Center Corporation Methods and compositions for programming an organic matrix for remodeling into a target tissue
US6649168B2 (en) 1999-03-17 2003-11-18 Novartis Ag Pharmaceutical compositions comprising TGF-beta
EP1113072A1 (de) * 1999-12-28 2001-07-04 Isotis B.V. Gewebetechnologie mit Zellen aus dem Kiefer
WO2001048152A1 (en) * 1999-12-28 2001-07-05 Isotis N.V. Tissue engineering using mandibular cells
WO2002032474A1 (en) * 2000-10-13 2002-04-25 Osteotech, Inc. Method for inducing new bone growth in porous bone sites
WO2002070023A2 (en) * 2001-03-05 2002-09-12 Hadasit Medical Research Services & Development Ltd. Compositions comprising bone marrow cells together with demineralized and/or mineralized bone matrix and uses thereof in the induction of bone and cartilage formation
WO2002070023A3 (en) * 2001-03-05 2004-02-19 Hadasit Med Res Service Compositions comprising bone marrow cells together with demineralized and/or mineralized bone matrix and uses thereof in the induction of bone and cartilage formation
WO2002069988A3 (en) * 2001-03-05 2004-02-26 Shimon Slavin Compositions comprising bone marrow cells together with demineralized and/or mineralized bone matrix and their use in bone marrow transplantation
WO2002069988A2 (en) * 2001-03-05 2002-09-12 Hadasit Med Res Service Compositions comprising bone marrow cells together with demineralized and/or mineralized bone matrix and their use in bone marrow transplantation
US7910554B2 (en) 2001-04-12 2011-03-22 Bioaxone Therapeutique Inc. Treatment of macular degeneration with ADP-ribosyl transferase fusion protein therapeutic compositions
WO2008022182A1 (en) * 2006-08-16 2008-02-21 The Uab Research Foundation Methods for promoting coupling between bone formation and resorption
USD849946S1 (en) 2015-12-30 2019-05-28 Nuvasive, Inc. Interspinous process spacer

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JPH06157339A (ja) 1994-06-03
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AU4468093A (en) 1994-02-24

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